The present invention relates to recombinant DNA which encodes the SfiI restriction endonuclease and modification methylase, and the production of these enzymes from the recombinant DNA.
Restriction endonucleases are a class of enzymes that occur naturally in bacteria. When they are purified away from other contaminating bacterial components, restriction endonucleases can be used in the laboratory to break DNA molecules into precise fragments. This property enables DNA molecules to be uniquely identified and to be fractioned into their constituent genes. Restriction endonucleases have proved to be indispensable tools in modern genetic research. They are the biochemical `scissors` by means of which genetic engineering and analysis is performed.
Restriction endonucleases act by recognizing and binding to particular sequences of nucleotides (the `recognition sequence`) along the DNA molecule. Once bound, they cleave the molecule within, or to one side of, the recognition sequence. Different restriction endonucleases have affinity for different recognition sequences. More than one hundred different restriction endonucleases have been identified among the many hundreds of bacterial species that have been examined to date.
Bacteria tend to possess at most only a small number restriction endonucleases per species. The endonucleases typically are named according to the bacteria from which they are derived. Thus, the species Haemophilus aegyptius, for example synthesizes 3 different restriction endonucleases, named HaeI, HaeII and HaeIII. Those enzymes recognize and cleave the sequences (AT)GGCC(AT), PuGCGCPy and GGCC, respectively. Escherichia coli RY13, on the other hand, synthesizes only one enzyme, EcoRI, which recognizes the sequence GAATTC.
While not wishing to be bound by theory, it is thought that in nature, restriction endonucleases play a protective role in the welfare of the bacterial cell. They enable bacteria to resist infection by foreign DNA molecules like viruses and plasmids that would otherwise destroy or parasitize them. They impart resistance by scanning the lengths of the infecting DNA molecule and cleaving them each time that the recognition sequence occurs. The breakup that takes place disables many of the infecting foreign DNA and renders that DNA susceptible to further degradation by non-specific endonucleases.
A second component of bacterial protective systems are the modification methylases. These enzymes are complementary to restriction endonucleases and they provide the means by which bacteria are able to protect their own DNA and distinguish it from foreign, infecting DNA. Modification methylases recognize and bind to the same nucleotide recognition sequence as the corresponding restriction endonuclease, but instead of breaking the DNA, they chemically modify one or other of the nucleotides within the sequence by the addition of a methyl group. Following methylation, the recognition sequence is no longer bound or cleaved by the restriction endonuclease. The DNA of a bacterial cell is always fully modified, by virtue of the activity of its modification methylase, and it is therefore completely insensitive to the presence of the endogenous restriction endonuclease. It is only unmodified, and therefore identifiably foreign, DNA that is sensitive to restriction endonuclease recognition and attack. Together, the restriction endonuclease and modification methylase make up what is commonly referred to as the restriction-modification system ("R-M system").
With the advent of genetic engineering technology, it is now possible to clone genes and to produce the proteins and enzymes that they encode in greater quantities than are obtainable by conventional purification techniques. The key to isolating clones of restriction endonuclease genes is to develop a simple and reliable method to identify such clones within complex `libraries`, i.e., populations of clones derived by `shotgun` procedures, when they occur at frequencies as low as 10.sup.-3 to 10.sup.-4. Preferably, the method should be selective, such that the unwanted, majority, of clones are destroyed while the desirable rare, clones survive.
Type II R-M systems are being cloned with increasing frequency. The first cloned systems used bacteriophage infection as a means of identifying or selecting restriction endonuclease clones (EcoRII: Kosykh et al., Molec. gen. Genet 178: 717-719, (1980); HhaII: Mann et al., Gene 3: 97-112, (1978); PstI: Walder et al., Proc. Nat. Acad. Sci. 78 1503-1507, (1981)). Since the presence of R-M systems in bacteria enable them to resist infection by bacteriophages, transformed host cells that carry cloned R-M genes can, in principle, be selectively isolated as survivors from libraries that have been exposed to phage. This method has been found, however, to have only limited value. Specifically, it has been found that cloned R-M genes do not always manifest sufficient phage resistance to confer selective survival.
Another cloning approach involves transferring systems initially characterized as plasmid-borne into E. coli cloning plasmids (EcoRV: Bougueleret et al., Nucl. Acid. res. 12: 3659-3676, (1984); PaeR7: Gingeras and Brooks, Proc. Natl. Acad. Sci. USA 80: 402-406, (1983); Theriault and Roy, Gene 19: 355-359 (1982); PvuII: Blumenthal et al., J. Bacteriol. 164: 501-509, (1985)).
A third approach, and one that is being used to clone a growing number of systems involves cloning by selection for an active methylase gene (See, e.g. EPO No. 193,413 published Sep. 3, 1986 and BsuRI: Kiss et al., Nucl. Acid. Res. 13: 6403-6421, (1985)). Briefly, methylase selection comprises screening for methylase clones by exposing DNA from transformed hosts with the corresponding restriction endonuclease. Survival indicates the presence of the methylase gene, presumably because the DNA of the host is modified and insensitive to attack by the restriction endonuclease. Since restriction and modification genes are often closely linked, both genes can often be cloned simultaneously. Methylase selection, however, does not always yield a complete restriction system however, but instead yields only the methylase gene (See, e.g., BspRI: Szomolanyli et al., Gene 10: 219-225, (1980); Bcn I: Janulaitis et al, Gene 20: 197-204 (1982); Bsu RI: Kiss and Baldauf, Gene 21: 111-119, (1983); and Msp I: Walder et al., J. Biol. Chem. 258: 1235-1241, (1983)), See also Wilson, Gene 74: 281-289, (1988); Slatko, et al., Gene 74: 45-50, (1988); Lunnen, et al., Gene 74: 25-32, (1988); VanCott, et al., Gene 74: 55-59, (1988).
There are, a number of possible explanations for such failures, and a variety of potential obstacles which the genetic engineer faces even in the methylase selection approach. In some systems, the cloning problem may lie in trying to introduce the endonuclease gene into a host not already protected by modification. If the methylase gene and endonuclease genes are introduced on a common DNA fragment, the methylase gene must modify or protect the host before the endonuclease gene cleaves the host's genome.
Another obstacle to cloning these systems in E. coli was discovered in the process of cloning diverse methylases. For example, many E. coli strains (including those normally used in cloning) have systems that resist the introduction of DNA containing methylation. (See, Raleigh and Wilson, Proc. Natl. Acad. Sci., USA 83: 9070-9074, (1986)). Therefore, it is also necessary to carefully consider which E. coli strain(s) to use for cloning.
Because highly purified restriction endonucleases, and to a lesser extent, modification methylases, are useful tools for characterizing and rearranging DNA in the laboratory, there is a commercial incentive to obtain strains of bacteria through recombinant DNA techniques that synthesize these enzymes in abundance. Such strains would be useful because they would simplify the task of purification as well as providing the means for production in commercially useful amounts.